Bone marrow endothelial progenitors in atherosclerotic plaque resolution

Abstract

Atherosclerosis is a major cause of morbidity and mortality in the United States. Persistently elevated circulating low-density lipoprotein, or hypercholesterolemia, and deposition of low-density lipoprotein in the vascular wall are the main inducers of atherosclerosis, which manifests itself as arterial lesions or plaques. Some plaques become thrombosis-prone and rupture, causing acute myocardial infarction or stroke. Lowering plasma cholesterol through the use of statins is the primary intervention against atherosclerosis. Treatment with statins slows progression of atherosclerosis but can only support limited plaque regression. Partially regressed plaques continue to pose a serious threat due to their remaining potential to rupture. Thus, new interventions inducing complete reversal of atherosclerosis are being sought. Implementation of new therapies will require clear understanding of the mechanisms driving plaque resolution. In this Commentary, we highlight the role of bone marrow endothelial progenitors in atherosclerotic plaque regression and discuss how regenerative cell-based interventions could be used in combination with plasma lipid-lowering to induce plaque reversal in order to prevent and/or reduce adverse cardiovascular events.

Introduction

Atherosclerosis is a major cause of death and disability globally. It is a chronic inflammatory disease of the arterial vascular wall triggered by dyslipidemia and endothelial distress. Deposition of low-density lipoprotein (LDL) in the sub-endothelium and the consequent pro-inflammatory reactions of cellular elements in the arterial wall promote influx of inflammatory leukocytes into the vascular wall and support the development of multifocal atherosclerotic plaques. Most plaques are asymptomatic and some become obstructive, but only a few become thrombosis-prone and may rupture, causing complications including acute myocardial infarction and stroke.^1,2

Statins are widely used to treat atherosclerosis. Large randomized clinical trials have documented their benefits in patients at risk for or presenting established atherosclerotic cardiovascular disease. Statins lower plasma cholesterol by reversibly inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis in the liver. They have also been shown in both experimental and clinical studies to have potent anti-inflammatory, vasodilatory, and anti-platelet effects that are independent of their lipid-lowering effects. These non-lipid-lowering effects depend on HMG-CoA reductase inhibition in tissues other than the liver. The effects include improvement of endothelial function, decreased vascular smooth muscle cell proliferation, attenuated vascular inflammation, increased plaque stability, and prevention of thrombus formation.³ Recent studies investigating effects of long-term intensive statin therapy on atherosclerosis burden have demonstrated that statins can also promote plaque regression. Regression, however, is incomplete, leaving patients prone to cardiovascular complications.^4,5 Thus, efforts are being made to develop new approaches that induce complete plaque resolution. Novel therapies will require a clear understanding of mechanisms driving plaque resolution that could be gained from pre-clinical animal models in which classic cardiovascular risk factors, especially dyslipidemia as the main inducer of plaque development, are reversible.

Mouse models of atherosclerotic plaque regression

Mouse models of atherosclerosis have proven valuable to uncover pathways of disease progression.⁶ Unfortunately, well-characterized atherosclerosis-prone mouse models such as low density lipoprotein receptor-deficient (Ldlr^−/−) and apolipoprotein E-deficient (ApoE^−/−) mice cannot be used for studies investigating plaque regression because genetic modifications in ApoE^−/− and Ldlr^−/− strains predispose these mice to irreversible diet-induced hyperlipidemia.⁷ Unlike in humans, statins do not evoke constant dose-dependent and uniform cholesterol-lowering in ApoE^−/− mice⁸ and have variable, weak effects on cholesterol levels in Ldlr^−/− mice.⁹ As reversal of hypercholesterolemia is mandatory for plaque regression,¹⁰ statin treatment of hyperlipidemic mice is an inadequate model to study atherosclerosis reversal.

As one experimental approach to this problem, adenovirus-mediated restoration of apolipoprotein E (ApoE) expression in ApoE^−/− mice effectively reverses hypercholesterolemia and reduces foamy macrophage burden in lesions. The main mechanism supporting regression of plaques in this model is reduced monocyte entry into lesions combined with a stable rate of macrophage apoptosis.¹¹ Regression of atherosclerosis occurs only under high level ApoE expression. This requirement is yet to be achieved in clinical settings.

Another strategy to study mechanisms supporting atherosclerosis regression is transplantation of plaque-containing portions of aorta from hypercholesterolemic ApoE^−/− mice into normolipidemic C57BL/6 recipients. In this model, normolipidemic environment in C57BL/6 mice enables plaque regression, which coincides with rapid emigration of inflammatory cells from lesions.¹² The experimental design in this investigation rapidly eliminates hypercholesterolemia and disrupts lymphatic and arterial systems, all of which do not mimic statin-induced changes in patients and may therefore limit translation of these findings to clinical settings.

Atherosclerotic plaque regression is also observed in the Reversa model (Ldlr^−/−ApoB^100/100Mttp^fl/flMx1-Cre, C57BL/6 background).¹³ Reversa mice lack low density lipoprotein receptor and express atherogenic apolipoprotein B 100. These genetic modifications make this strain susceptible to atherosclerosis. Thus, high fat diet-induced hyperlipidemia causes aortic atherosclerosis reminiscent of that in ApoE^−/− and Ldlr^−/− strains. However, hypercholesterolemia can be reversed upon Cre-dependent inactivation of the microsomal triglyceride transfer protein (Mttp), which is required for the transport of neutral lipids to nascent ApoB lipoproteins and the assembly of atherogenic LDL in the liver.¹⁴ The Reversa model is ideal for studying regression of atherosclerosis because genetic inactivation of Mttp results in moderate and time-dependent lowering of plasma lipids that mimics statin-induced normalization of plasma lipids in patients.

Endothelial progenitor cells in cardiovascular disease

The major event initiating atherosclerotic plaque development is hypercholesterolemia-induced endothelial dysfunction. The endothelium is a key constituent of the vascular wall that is actively involved in maintaining the integrity and proper functioning of blood vessels. Endothelial dysfunction changes endothelial cell morphology, intercellular junctions, oxidative and inflammatory status, nitric oxide homeostasis, lipoprotein transport, and anti-thrombotic properties. All of these alterations promote plaque development and contribute to clinical manifestations of atherosclerosis.¹⁵ Thus, it is not unreasonable to suggest that normalization of plasma lipids and vascular repair supported by endothelial regeneration may be required for atherosclerosis to regress. The main cell type supporting regeneration/repair of vascular endothelium is endothelial progenitor cells (EPCs).¹⁶

EPCs were first described by Asahara et al. in 1997 as peripheral blood cells expressing both CD34 and vascular endothelial growth factor receptor 2 (VEGFR2 or Flk1 in the mouse) that stimulate vascular repair in vivo.¹⁶ Based on this definition, the main approach to identify EPCs is to identify cells bearing surface markers that indicate both cellular immaturity (Sca-1, c-kit, CD133, CD34) and endothelial origin (VEGFR2/Flk1). As the antigenic combination is not fully specific for EPCs, these cells may also be identified as cells that uptake 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL (Dil-Ac-LDL) and/or bind lectin.¹⁷

EPCs can have hematopoietic or non-hematopoietic origin. Hematopoietic EPCs, derived from bone marrow, are a heterogeneous population represented by colony-forming EPCs, non-colony-forming differentiating EPCs, myeloid EPCs, and angiogenic cells. These cells are mainly found in circulation. Non-hematopoietic EPCs can be isolated from blood or tissue samples. The origin of non-hematopoietic EPCs is unclear.¹⁸ Because of the rarity of EPCs and difficulties in identification, limited information is available about functional characteristics of EPC subtypes.

EPCs support vascular repair by homing to and differentiating into mature CD31⁺ endothelial cells in damaged endothelium. By so doing, EPCs promote vasculogenesis that directly contributes to endothelial regeneration. EPCs may also indirectly contribute to the re-establishment of endothelial homeostasis by producing pro-angiogenic cytokines and growth factors that promote proliferation of existing resident endothelial cells.¹⁸

EPCs were demonstrated to exert beneficial effects in ischemic conditions in rodents and human.¹⁹ However, their role in atherosclerosis remains controversial. Hypercholesterolemia reduces circulating EPCs and may deplete EPC bone marrow pool. The functional characteristics of EPCs, such as proliferation, migration, and vasculogenic potential, are also impaired in hypercholesterolemic patients. Thus, hypercholesterolemia limits EPC homing and regenerative potential. Reciprocally, correction of hyperlipidemia correlates with an increase in circulating EPCs and improved cardiovascular risk profiles.²⁰

Treatment with EPC mobilizing agents such as granulocyte colony stimulating factor (G-CSF) prevents progression of atherosclerosis and improves symptoms of intractable atherosclerotic peripheral artery disease in patients.²¹ Interestingly, accelerated plaque development was observed following administration of EPC mobilizer vascular endothelial growth factor (VEGF) into hypercholesterolemic ApoE^−/− mice.²² Furthermore, investigations measuring the impact of cell-based therapy on plaque development, especially infusion of a pure EPC population, have also generated inconsistent results. Chronic treatment with bone marrow EPCs from non-atherosclerotic young ApoE^−/− mice prevented atherosclerosis progression in adult ApoE^−/− recipients despite persistent hypercholesterolemia.²³ In contrast, administration of spleen-derived EPCs resulted in an increase in atherosclerosis burden in ApoE^−/− mice.²⁴ Thus, EPCs, depending on their source (bone marrow vs. spleen) and the disease stage (early vs. late atherosclerosis), exert a variety of effects in an atherosclerotic milieu. Because atherosclerosis-prone mouse models are hypercholesterolemic and since hypercholesterolemia reduces recruitment, decreases survival, and limits functions of EPCs, the exact roles of these cells in atherosclerosis progression remain very limited. There have been no studies investigating contributions of EPCs to possible repair of vascular wall damaged by plaque development in conditions that do not limit their recruitment and regenerative potential. Thus, we have conducted such an investigation in Reversa mice, the mouse model in which hypercholesterolemia, the risk factor affecting EPC survival and regenerative potential, is reversible.

Bone marrow endothelial progenitor cells augment atherosclerotic plaque reversal

In Reversa mice, high fat diet induces hypercholesterolemia which triggers development of atherosclerotic plaques. Normalization of plasma lipids supports plaque regression in this model; however, atherosclerosis resolution is incomplete.¹³ Our investigation demonstrates that treatment of Reversa recipients in which hypercholesterolemia was normalized (atheroregressing mice) with bone marrow EPCs significantly reduces plaque burden, indicating that EPCs augment atherosclerosis regression. Furthermore, accelerated plaque resolution correlates with engraftment of bone marrow cells into endothelium and differentiation of endothelial progenitors to CD31⁺ endothelial cells, suggesting that EPCs are vasculogenic in atheroregressing environment. Importantly, we show that EPC treatment of regressing recipients increases atheroprotective nitric oxide in blood and improves vascular relaxation(Fig. 1).²⁵ Because these two parameters are important indicators of vascular health, our study shows that bone marrow EPCs are beneficial in regressing conditions as they reduce atherosclerosis burden and restore vascular function.

Figure 1. Endothelial progenitors in plaque reversal. In cardiovascular patients normalization of plasma lipids supported by statins, exercise and/or diet mobilizes EPCs (green) from internal stores such as bone marrow to peripheral blood. However, it remains unclear whether circulating EPCs contribute to repair of vascular wall damaged by plaque development in regressing conditions. Our study conducted in the Reversa mouse model shows that bone marrow endothelial progenitors reduce atherosclerosis burden thereby advancing plaque reversal. The accelerated plaque resolution coincides with incorporation of bone marrow EPCs into endothelium and differentiation of endothelial progenitors to mature endothelial cells. Importantly, EPC treatment of regressing Reversa recipients increases atheroprotective nitric oxide (NO) in blood and improves vascular relaxation.²⁵ Since treatment with EPCs reduces plaque burden and improves vascular function, our study clearly shows that bone marrow endothelial progenitors are pivotal in atherosclerosis resolution.

EPCs have not been evaluated for their potential to support regression of atherosclerosis in cardiovascular patients. Our finding, however, suggests that augmented regression of atherosclerosis could be achieved by an EPC-based approach. Thus, this study constitutes a foundation for the use of adult stem cells in combination with statins to treat atherosclerosis.

EPCs to treat atherosclerosis?

The main limitation in using EPCs to augment plaque resolution would be their low availability in peripheral blood of cardiovascular patients. Lipid lowering with statins,²⁶ diet or exercise²⁷ increases circulating EPCs ; however, it remains unclear whether this increase in EPC numbers is indeed sufficient to interfere with atherosclerosis progression, improve plaque stability, or facilitate disease regression. Our study showed that treatment of atheroregressing mice with bone marrow progenitor mobilizing agent AMD3100 (FDA-approved Mozobil) reduces atherosclerosis burden to the same extent as adoptive transfer of EPCs.²⁵ This suggests that release of EPCs from internal stores, especially from bone marrow, may be an intervention used to increase circulating EPCs. In addition to AMD3100, the other bone marrow progenitor cell mobilizer that may be considered is G-CSF. G-CSF has been used clinically for mobilization of hematopoietic stem cells (HSC) for over a decade. In addition to HSCs, G-CSF also mobilizes EPCs. G-CSF is approved for long-term use in patients with severe chronic neutropenia.²⁸ Adverse events associated with clinical use of G-CSF are not troublesome and seldom necessitate stopping therapy.²⁹ Mozobil is a more potent stem cell mobilizer than G-CSF³⁰; however, it is clinically approved only for acute and not chronic use. Both, Mozobil and G-CSF should be tested as it remains unclear whether Mozobil-induced substantial and short-term increase in circulating EPCs or moderate and long-term increase in EPC availability supported by G-CSF may be needed to advance atherosclerosis regression in cardiovascular patients treated with lipid-lowering drugs. Instead of mobilizing EPCs from bone marrow, administration of autologous EPCs harvested from peripheral blood by leukapheresis could serve as another therapeutic approach to increase EPC numbers in circulation in order to advance plaque resolution.

Conclusion

Complete resolution of atherosclerosis is a desired clinical goal; however, mechanisms supporting plaque reversal remain obscure. Our finding that normalization of plasma lipids in the experimental model of atherosclerosis regression, the Reversa mouse, leads to incomplete plaque regression which can be augmented by adoptive transfer of bone marrow endothelial progenitor cells, suggests that EPCs are pivotal in atherosclerosis resolution. Although further studies are needed to identify mechanisms supporting homing of EPCs to regressing plaques and to elucidate pathways by which endothelial progenitors repair vascular wall damaged by plaque development, the study forms a foundation for the use of adult stem cell-based approaches in development of new clinical interventions that would advance plaque regression to prevent and/or reduce lethal complications of atherosclerosis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.